Method for externally treating the blood

ABSTRACT

A method and system are disclosed for externally treating human blood, with the objective of reducing the functioning lymphocyte population in the blood system of a human subject. According to the method, blood is withdrawn from the subject and passed throught an ultraviolet radiation field in the presence of from about 1 nanogram to 100 micrograms per ml of blood, of a dissolved photoactive agent capable of forming photo-adducts with lymphocytic-DNA, mobile cortisone receptors or antigen sites to thereby effect covalent bonding between the photoactive agent and the same, thereby inhibiting the metabolic processes of the lymphocytes or complexing them; and thereupon returning the irradiated blood to the subject. The withdrawn blood may be formed into an extracorporeal stream and flowed through a treatment station whereat the irradiation is effected, as for example by exposure to UV radiation; and such flow process may be conducted on a continuous basis. If desired, at least portions of the treated blood may then be separated, as for example by a continuous centrifuge, before returning the remaining diverted blood to the subject.

This is a division of application Ser. No. 272,981 filed June 12, 1981now U.S. Pat. No. 4,398,906, which in turn is a continuation-in-part ofapplication Ser. No. 102,553, filed Dec. 11, 1979, now U.S. Pat. No.4,321,919.

This invention relates generally to a method for medical treatment of aliving mammal, and more specifically relates to a method for treatingthe blood supply of a living subject with photoactive chemical agentswhich when activated form photoadducts with blood constituents for thepurpose of reducing the functioning population of those constituents inthe blood supply of the subject.

The method of this invention has particular applicability in a number ofhighly significant human diseases, including certain forms of leukemia,where the population of certain types of leucocytes, includingespecially lymphocytes, increases inordinately in comparison to theother populations of nucleated cells in normal blood. While theexcessive population of such lymphocytes represents a result of, ratherthan the underlying cause of the disease, the excessive lymphocytepopulation brings direct adverse effects to the patient if steps are nottaken to reduce same. Complications thus rapidly develop which impairthe functioning of bodily organs, and eventually a life-threateningsituation is presented.

It should also be appreciated that excessive increase in the lymphocytepopulation of the blood supply can occur in other human maladies, inaddition to lymphocytic leukemias. Thus, for example, such results canobtain in consequence of severe allergic reactions to administeredagents, including drugs or the like, or in many otherlymphocyte-mediated diseases.

In addition to the development over the years of pharmaceutical agentsand the like, which may nonspecifically reduce the lymphocytepopulation, e.g. by altering the underlying production rate of same,various techniques have from time to time been used in an effort todirectly attack the problem, as for example by mechanically removingsuch lymphocytes from the blood supply. It is thus known, for example,to pass the blood supply through a continuous centrifuge, whereat oneseeks to selectively remove lymphocytes to reduce the population of thelatter in the thereby processed blood supply. In general, however, thismethod tends to be very inefficient, in part because the densitydifferences between the blood fractions including the undesiredlymphocytes and fractions which include desired blood components, isinsufficient to assure that high percentages of the former are removedwhile retaining high proportions of the latter.

It is also well-known to treat diseases such as leukemia with highenergy electromagnetic radiation, including in the x-ray region. Whilesuch treatment is often directed at internal bodily organs whereat theblood cells are being generated, it has also been known to irradiate theblood supply with x-radiation at a point external to the body (the bloodhaving first been withdrawn), whereby the radiation is not rendereddirectly incident on the body or internal organs of same. This method,while powerful, is indiscrimate, in that the intensely disruptiveenergy, in addition to destroying undesirable cells, disables ordestroys components of the blood which are desired to be retained invital status.

Among the pharmaceutical agents used to treat the excessive lymphocytepopulation resulting from leukemia are agents which are active againstthe lymphocyte itself. Cortisone is one such agent, its effectiveness,however, is limited as it does not completely suppress the aberrantmetabolic activity of the malignant lymphocyte. The mechanism by whichcortisone acts on lymphocyte cells is not fully understood, it isbelieved, however to initially bind specifically to the cortisonereceptors in the lymphocyte and to be carried by these mobile receptorsto the cell's nucleus wherein it acts to alter the metabolic activity ofthe cell.

Certain other chemical agents are known or are believed to weakly bindto the nucleic acids of certain nucleated cells where they intercalateby forming molecular complexes involving low energy chemicalinteractions or intermolecular attractions, which generally aretransient and insufficient to significantly affect the rate of DNAsynthesis in the cell. The "psoralens" which are described in my earlierfiled application, Ser. No. 102,553, of which this application is acontinuation-in-part, are such chemicals.

Certain ligating proteins, known as antibodies, are also active againstlymphocytes. The interaction of an antibody with a particular lymphocyterequires that the lymphocyte have a site or antigen which isgeometrically and chemically receptive to a corresponding active site onthe antibody. The forces which bind an antibody to an antigen consist ofattractive forces including hydrogen bonding, apolar bonding, ionicinteractions and Van der Waals interactions, the strength of which areinversely proportional to the distance between the interacting groups.Accordingly, any structural variations in the lymphocyte membrane whichserve to alter the geometry of the antigen can serve to prevent thebinding of an antibody to the antigen. Further, once an antibody bindsto an antigen on a cell, the cell may undergo "antigenic modulation" oraltered cellular differentiation and thereby break the antibodies' bondon it and destroy the antibodies' affinity towards it. Where alymphocyte's membrane has a structure which blocks the antibody from itsantigenic site, the antibody while still attracted to the antigen willbe unable to form any linkage of a permanent nature. Inasmuch asvariations in cell structure are more the rule than exception withmalignant cells, and "antigenic modulation" occurs in a high percentageof the antibody-cell antigen couplings, it has not been possible toeffectively combat leukemia cells with antibodies.

The use of antibodies to permanently inactivate or remove immunogenicchemicals which may be found in the blood, such as undesirable naturalantibodies, has also been hindered by the inability of an antibody toirreversibly complex with antigens.

The above-described pharmacologic interactions can be strengthened byuse of photoactive chemical analogues. Photoactive chemical agents arecompounds containing one or more groups which are excited by incidentultraviolet radiation and which when activated have a tendency to formcovalent linkages with nearby chemical groups. The reactivity of variousphotoactive agents varies from the chemically specific, which is thecase with agents such as the psoralens, to agents having greatreactivity toward virtually any group, which is the case with diazoesand azides. The diazoes and azides are the preferred photoactivemoieties for inbuing chemical agents to be used in the invention withphotoactivity which is essential in the method of the invention.

Until the present invention, photoactive chemical agents have beenutilized only in very limited fashions. On a clinical level, one classof photoactive compounds, the psoralens, have been used to treatpatients suffering from psoriasis. Other uses of these agents have beenalmost exclusively experimental investigations of cell physiology andchemistry, typical reports of which appear in the following articlesappearing in the Annals of N.Y. Acad. Sci. 346, "Photoaffinity Probes inthe Antibody Combining Region", Richards, F. F. and Lifter, J., pp.78-89; and "Photolabile Antibiotics As Probes of Ribosomal Structure andFunction", Cooperman, B. S., pp. 302-323.

SUMMARY OF THE INVENTION

Now, in accordance with the present invention, a method has been foundwhich enables safe and effective reduction of the functioning populationof certain blood constituents. More particularly, the method of thepresent invention enables the reduction of the functioning population ofcertain nucleated cells and undesirable antigenic chemical substances,such as undesirable auto-reactive antibodies, in the blood supply of ahuman subject.

According to the method of invention, blood requiring such treatment iswithdrawn from the subject and irradiated with UV radiation in thewavelength range of from about 2000 to 4000 Angstroms, and preferablyfrom about 3200 to 4000 Angstrons (UVA), in the presence of an effetiveamount of a dissolved photoactive chemical agent of the type capable ofintermolecular or chemical association with:

(1) the nucleic acids of nucleated blood cells,

(2) the steroid receptor sites of nucleated blood cells,

(3) the antigenic sites on nucleated blood cells,

(4) or the antigenic sites on immunogenic chemicals.

Upon irradiation, the photoactive chemical agent is induced to form apermanent photo-adduct with its associated site in or on the nucleatedblood cell or immunogenic chemical whereby the destruction of theadducted constituent is assured. The irradiated blood is then returnedto the subject.

When a photoactive chemical agent having an affinity for the nucleicacid of nucleated cells, such as lymphocytes, is employed in the presentinvention, the aforementioned intermolecular attractive forces draw theagent into an intercalated relationship with the nucleic acids of thelymphocytes. Prior to activation, the agent has little or no effect onthe cell chemistry, however, upon irradiation the agent forms certaincovalent attachments with the nucleic acids of the cell and therebyinhibits the metabolic functions of the cell. In this fashion, thecell's processes having been disrupted, and in particular its ability todivided, the death of the cell results.

The family of chemicals known in the art as the psoralens and more fullydescribed in my copending application, Ser. No. 102,553, filed Dec. 11,1979, the disclosure of which is incorporated herein by reference, havebeen found to have the activity described herein and are deemed wellsuited for application in the present invention. Photoactive chemicalagents, having an affinity for DNA, such as the psoralens, when used inthe invention have a highly desirable benefit in that the impairment anddestruction of lymphocytes tends to be selective in certain diseasessuch as leukemia to the cells most sought to be reduced, by virtue ofthe fact that it is such cells which are undergoing the most intensemetabolic activities to begin with, whereby they are the cells mostsubject to disablement by their present process.

Cortisone is a chemical agent having an affinity for particularreceptors within the nucleated lymphocyte cell. As has been previouslyindicated, cortisone's applications in reducing the functioninglymphocyte population in patients suffering from leukemia have been lessthan entirely satisfactory. According to the present invention, however,cortisone can be utilized to treat leukemia in a new and far moreeffective fashion.

Prior to application in the invention, cortisone must first be renderedphotoactive. Those skilled in the art will appreciate that thephotoactivation of cortisone can be achieved using established chemicaltechniques. The particulars of that chemistry are not deemed to bewithin the scope of this invention, which is limited to a method wherebycertain chemical agents can be employed to achieve previouslyunattainable reductions in functioning population of certain bloodconstituents. Those skilled in the art will also recognize thatemploying established chemical procedures, including where necessarythat of binding site protection, cortisone can be infused with aphotoactive moiety at several positions and that the substitutedcortisones can be evaluated and the homologue retaining the largestpercentage of cortisone's normal biological activity easily determined.The chemistry of the aforedescribed photoactivation and determination ofmost active homologue is thorougly discussed in the following articles:

(1) Katzenellenbogen, J. A., H. N. Myers and H. J. Johnson, Jr., 1973,J. Org. Chem. 38: 3525-33.

(2) Katzenellenbogen, J. A., H. J. Johnson, Jr. and H. N. Myers, 1973,Biochemistry 12: 4085-92.

(3) Katzenellenbogen, J. A., H. J. Johnson, Jr., K. E. Carlson and H. N.Myers, 1974, Biochemistry 13: 2896-94.

As these articles disclose, the photoactivation of steroids has beenachieved with great success through the substitution of the photoactivemoieties known as diazo and azide groups. These groups individually havea high degree of intrinsic photoactivity and that activity is retainedwhen they are incorporated into another chemical agent, therebyrendering it photoactive.

Employing then, known techniques of photoderivatization, the16-diazocortisone, which is preferred in the invention for retaining ahigh degree of its original pharmacological activity, can be synthesizedin good yield by first nitrosating cortisone to give 16-oximocortisone,which can be converted into the 16-diazocortisone by chloramineoxidation. Other substituted cortisones which may be derived by thenitration of cortisone using nitric acid in glacial acetic. The productsof this nitration step are a number of azide derivatives, which caneasily be separated by column chromatography. The reaction parametersemployed to make these products can be found in enabling detail in theaforementioned Katzenellenbogen article in the J. Org. Chem. 38:3525-33.

Photo-derivatized cortisone, having the preferred structure disclosedabove or one of the other possible less preferred homologues, uponaddition to the blood, readily enters the lymphocytes or other nucleatedcells and associates itself with the cortisone receptor sites in thosecells. After a suitable interval, calculated to allow a high percentageof the substituted cortisone to reach these receptor sites, typically inthe range of 1 minute to 2 hours, and preferably 5-15 minutes, the bloodcontaining from about 1 nanogram to 100 micrograms of dissolvedphotoactivated cortisone is irradiated with UV radiation. Irradiation ofthe blood activates the photoactive moiety on the cortisone molecules insitu at the cortisone receptor sites and causes the formation ofphoto-adducts between the substituted cortisone and the cortisonereceptor, as a consequence of which the receptors' ability to transmitcortisone vital to the continued metabolic activity of the cell isdestroyed. Accordingly, a very large fraction of the cortisone receptorsin the lymphocytes having been inactivated, the cells quickly becomeunable to function, and most particularly to divide, and theirdestruction rapidly follows.

Antibodies specific to particular blood constituents can be generatedbut, as has been indicated, it has not been possible to employ them withgood results in reducing the population of malignant cells in the bloodbecause of the variations in structure which are common with malignantcells and cellular phenomena such as antigenic modulation which enablesa cell to rid itself of a complexed antibody. Thus, for example,antibodies specific for a particular type of malignant T-lymphocyte maybe unable to complex with a large fraction of the cells of that type inthe blood, regardless of the antibodies having an affinity toward thosecells, and a significant number of lymphocytes having been complexed bythe antibodies shed their bound antigens breaking the antibodies' holdon them. According to the present invention, however, photoactivatedantibodies can be utilized to reduce the functioning lymphocytepopulation to a previously unobtainable degree. Moreover, employing themethod of the present invention photoactivated antibodies specificallyreactive to other blood constituents such as, undesirable antibodies,can also be employed to reduce the population of those constituents inthe blood with similar great effectiveness.

The methods whereby an antibody specific for a particular cell orimmunogenic chemical may be produced and purified are well known in theart and need not be recited here, suffice it to say that largequantities of very specific monoclonal antibodies can be made byHybridoma or by other established techniques.

The chemical techniques whereby an antibody desired for use in thepresent invention may be rendered photoactive are also well known tothose skilled in the art. It will also be obvious to those skilled inthe art, that virtually all antibodies have a number of sites suitablefor photoactive derivatization.

Methods whereby moieties foreign to an antibody may be added theretowithout injurying the antibodies' ability to complex with its specificantigens, for example, have been disclosed in the Handbook ofExperimental Immunology, Weir, D. M., pub. J. B. Lippincott, 1978, pp.15.1-15.30. In furtherance of the objective of this invention, it isimportant that the process of derivatization not destroy the combiningregion of the antibody which is specific for the target cell. In thisregard, it should be noted that in view of the number of potential sitesfor derivatization on most antibodies and the many different techniqueswhereby they may be infused with a photoactive moiety, such as thepreferred diazo and azide groups, it will rarely be necessary to takethe precaution of specifically protecting the combining region. Where,however, it is found that the combining region on an antibody wouldotherwise be destroyed by the photoderivatization of that antibody,established techniques of combining site protection and subsequentremoval of the protecting group can be employed.

When photoactivated antibodies specifically reactive to some bloodconstituent, which for example in a preferred embodiment of thisinvention might be the malignant T-lymphocytes of a patient, are addedto that patient's blood in the method of the present invention, theywill very quickly complex with T-lymphocytes for which there exists therequired correspondence of antibody combining region and cell antigenicsite. Numerous antibodies, however, by reason of deficiences in theirown combining regions or in their target cells' receptors, whileattracted to the target lymphocytes will be unable to form a trueantibody-antigen complex. Upon irradiation with UV radiation, of awavelength capable of activating the particular photoactive moiety whichhas been infused into the antibodies, the photoactive moieties on theantibodies which have complexed will perferentially form photo-adductswith the complexed cells, thereby permanently binding them to theircomplexed antibody and eliminating the possibility of antigenicmodulation. Other antibodies which had previously failed to complex withany of the target cells to which they were attracted because ofinsufficient correspondence in their respective binding regions, willupon photoactivation form photo-adducts with those cells, therebycreating a photo-adduct complex where none had existed before. In thedescribed fashion, an antibody complexing effectiveness never beforethought possible is achieved.

The aforedescribed photo-induced antibody-antigen complexing isamplified by the infusion of multiple photoactive groups into theantibody structure, for the presence of several photoactive moieties onthe antibody increases the likelihood that a single antibody will beable to complex with more than one target cell. When such antibodies areemployed according to the method of the invention, they have an enhancedtendency to form networks or chains of complexed cells which can beremoved from the blood with particular facility.

It is also within the scope of this invention that antibodies specificto particular undesirable natural antibodies or other immunogenicchemicals can be rendered photoactive and used according to the methodof the invention to form irreversible complexes which will enable thosechemicals to be removed from the body with a far greater efficiency thanpreviously possible.

According to the method of the invention regardless of the type ofphotoactive chemical agent employed therein, blood withdrawn from asubject for treatment can be handled in batch form, but preferably isformed into an extracorporeal stream and passed through a treatmentstation whereat the irradiation is effected. Such a treatment stationmay take the form of an extended flattened tubular passageway, the wallsof which are substantially transparent to the incident ultraviolet light(UV) used to activate the photoactive chemical agent. Typical radiationdoses range from about 0.1 to 100 joules per cm² and preferably fromabout 5 to 60 joules per cm² of blood surface whether the process iscarried out on a continuous or discontinuous bases, and typical flowrates through the irradiation station can be in the range of from about10 to 75 m./min.

Following treatment, the entire batch, or irradiated flow of divertedblood, can be returned to the patient. However, depending on whichphotoactive chemical agent was employed in the treatment of the blood,it may be preferable to filter or centrifuge the treated blood prior toits return to the patient. The instances in which such treatement wouldbe deemed appropriated are more fully elucidated in the detaileddescription of this invention.

BRIEF DESCRIPTION OF DRAWINGS

The invention is diagrammatically illustrated, by way of example, in thedrawings appended hereto, in which:

FIG. 1 is a schematic flow diagram illustrating a preferred embodimentof a system operating in accordance with the present invention;

FIG. 2 is a schematic elevational view of the irradiation stationportion of the FIG. 1 system;

FIG. 3 is a plan view, schematic in nature, of one embodiment of theirradiation station of FIG. 2; and

FIGS. 4 and 5 are cross-sectional views, taken along the lines 4--4 and5--5 of FIG. 3, and illustrate the configurations of the flow passagewayand the output passage for the FIG. 3 device.

DESCRIPTION OF PREFERRED EMBODIMENT

In FIG. 1 herein a schematic diagram appears of a system 10 inaccordance with the present invention. Except for the irradiationstation, the bulk of the components of system 10 are per se conventionaland known in the art; and hence it is not deemed appropriate ornecessary to vastly detail same.

As indicated in the Figure, blood may initially be withdrawn from thehuman subject, as at 12. Typically the blood is withdrawn via a donorneedle, which may e.g. be emplaced at the right antecubital vein. In theshowing of FIG. 1, it is assumed that the processing of blood pursuantto the invention is conducted on a continuous basis, i.e. for purposesof the present discussion the flow may be regarded as continuous fromwithdrawal at 12, to final return of the blood to the subject at 14.Such return 14 is typically effected via a recipient needle positionedin the left antecubital vein. Where the flow is indeed continuous inthis manner, a typical blood flow utilizable in practice of theinvention is in range of from about 10 to 75 ml/min. with a morepreferred range being from about 40 to 50 ml/min. The indicated flowrates are effected by means of a pump 16, which is positioned in theextracorporeal blood flow stream generally indicated at 18, and maycomprise one of numerous types of pumps used for blood flow treatmentpurposes, including such pumps as those available from Haemonetics Corp.under Model Designation 30.

As is known in the pertinent medical art, anti-coagulants are preferablyinjected into the extracorporeal blood flow stream at 20, i.e. close tothe point of blood withdrawal. Such anti-coagulants can comprisesolutions of acid citrate dextrose and/or of heparin, or of other knowncompositions useful for this purpose.

An occluded vein sensor 22 is preferably provided in stream 18 forpurposes, as known in the art. Such sensor basically comprises areservoir or buffer volume, the object of which is to prevent or inhibitgeneration or continued existence of bubbles in the blood flow stream.

Pursuant to a preferred mode of practicing the present invention, thephotoactive chemical agent is preferably added to the blood of the humansubject external to such subject; and thus as shown in the system 10 ofFIG. 1, may be provided to the flowing blood downstream of pump 16, andjust upstream of where the blood enters the irradiation station 24.

As has been discussed under the "Summary of Invention", the preferredphotoactive chemical agents for use in the process of the invention arethe psoralens, photoactivated cortisone, photoactivated antibodiesspecfically reactive to malignant lymphocytes and photoactivatedantibodies specifically reactive to a patient's undesirable antibodies.As was also indicated, other photoactive chemical agents, are alsoutilizable in the method of the invention. The basic technique used inintroducing photoactive chemical agents, is to dissolve same in anisotonic solution, which thereafter is directly injected into theflowing blood streams, as at 26. The agents are injected at a rate incomparison to the blood flow rate as to achieve a concentration in theblood thereafter passed to irradiation station 24 in the desired range,described for each of the chemical agents of the invention.

In the foregoing connection it should be appreciated that the primaryobjective of the operations thus far described is one of achieving thedesired dissolved concentration of the photoactive chemical agent priorto introduction of the blood to the irradiation station. In accordancewith a further aspect of the invention, it will therefore be appreciatedthat the said photoactive agent need not necessarily be directlyintroduced by injection into the extracorporeal blood stream 18 flowingin FIG. 1. Rather, it is also acceptable to achieve the desiredconcentration of photoactive agent by orally or otherwise administeringthe compound directly to the patient. Where, for example pursuant to theinvention, psoralen is orally administered, it can be provided in oraldosages of from about 0.6 to 1.0 mg per kg of body weight. The desiredconcentration range in the blood used for practice of the invention, isthen achieved in about two hours from oral administration. Alternatemodes of administration for the other photoactive chemical agents withinthe scope of this invention and the doses appropriate therefor will beapparent to those skilled in the art.

However, it is preferred to introduce the photoactive chemical agents ofthe invention to the extracorporeal stream (or to an extracorporealbatch volume) in order to achieve more exact concentration levels; andfurther, to avoid or minimize possible side effects and the like, whichcan occur from administration of any drug directly to the body system.

At irradiation station 24, consisting of an irradiation chamber 28 andradiation source 30, the blood now carrying in solution the desiredconcentration of photoactive chemical agent, is subjected to ultravioletradiation (UV) and preferably UV radiation having the bulk of itsspectral components in the preferred range for the activation of theparticular photoactive agent being employed in the treatment beingconducted. The materials of construction of the irradiation station 24are selected so as not to block radiation in the desired position of theUV spectrum.

In FIG. 2, a schematic elevational view appears of an irradiationstation 24 of a type suitable for use with the invention. Such stationconsists of a blood treatment or irradiation chamber 28, having an inlet31 and an outlet 32, enabling blood flow through the chamber, and aspaced source 30 or UV radiation. The chamber 28 can take various forms,with the principle requirement for same being that the wall 34 of sameopposed to source 30, be substantially transparent to the incident UVradiation. The said chamber (or at least wall 34) can thereforetypically be comprised of various substantially UV-transparent plastics,as are commonly used in tubing constructed for administration ofstandard intravenous solutions, such as polyvinyl chloride and the like.

In one embodiment of chamber 28, the said device can comprise a simpleenvelope, i.e., the central void 36, is substantially of thinrectangular cross-section. Where, however, the blood is to be treated aspreferred, on a continuous basis, superior flow characteristics andbetter control of the exposure time can be achieved where bloodtreatment chamber 28 has a configuration as shown in FIGS. 3, 4 and 5.In this instance a tubular coil 38, which in cross-section (FIG. 5) isflattened to a very elongated elipse, is fixedly maintained in or upon asupport plate 40. The blood flow inlet 30 to the coil is of circularcross section, and in terms of FIG. 1 is at a point downstream of pump16. The feed-in for the photoactive chemical agent is schematicallydepicted at 26. The highly flattened cross-section of the coil enablesgood flow for the blood passing through the coil, but more importantly,enables good exposure of the flowing blood to the incident UV radiation.The outlet 32 is again returned to a circular cross-section.

Regardless of the design selected for the chamber 28, it is preferredthat the chamber be as thin as practicable. Chambers having a thicknessin the range of 0.05 to 10 mm are within the range contemplated in theinvention, with chamber thicknesses in the range of about 0.05 mm to 1mm preferred.

UV source 30 may comprise one or a plurality of side-by-side orotherwise arranged UV light sources 41, each of which may be backed by areflector 42. The UV sources can comprise commercially available lamps,numerous types of which are known in the art.

By way of example, source 30 can comprise a single 1000 watt Hg lamp ofthe type available from Oriel Corporation of Stamford, Conn., underModel designation 6287. When used with appropriate filters this sourceprovides a good relatively continuous spectrum of high intensityradiation between 3200 and 4000 Angstroms, with a peak emission at about3650 Angstroms, which is preferred when psoralen is the photoactiveagent being employed in the method of the invention. The said lamp witha suitable reflector can be positioned approximately 5 to 30 cm fromchamber 28. With the flow rates utilized in accordance with one aspectof the invention, such a source will provide absorbed energy in theflowing blood within the range of interest for practicing the method ofthe invention.

The blood flow from irradiation station 24 proceeding as shown in FIG. 1via outlet 32, can be directly returned to the subject at 14.Optionally, however, prior to returning the treated blood to thepatient, it may be heat exchanged so as to adjust its temperature tothat of the patient's circulating blood. Heat exchange as described isnecessary whenever the treated blood, by consequence of its treatment,has attained a temperature substantially at variance with that of thepatient.

Where the method of the invention has been employed to reduce thefunctioning lymphocyte population of the blood, employing either a DNAactive agent, such as a psoralen, or a photoactivated cortisone, thetreated lymphocytes upon return to the patient as a consequence of theirtreatment will be rapidly broken down and destroyed by the normalprocesses occuring in the patient. More specifically, by their treatmentaccording to the aforementioned embodiments of the invention, themetabolic functions of the treated lymphocytes are impaired to theextent that with appropriate doses of photoactive agent and UV radiationa substantial percentage of the treated cells will be destroyed on agradual basis over a period of days. A benefit of this feature of theinvention is that it is thus possible to treat substantially the entireblood supply of a patient in a single treatment without causing thecatastrophic overloading of the body's blood purification system whichwould otherwise result if the entire population of treated lymphocyteswere to succumb to the treatment at the same time.

Where photoactivated antibodies specific to a malignant lymphocyte areemployed in the other preferred embodiments of the invention, the bloodreturned to the subject will have its lymphocytes (or alternatively anundesirable antibody) complexed by the activated antibody and thustagged for removal from the blood stream. Since, however, thephotoactivated antibody complexes formed according to this embodiment ofthe invention are essentially completely formed prior to the exiting ofthe blood from the irradiation station 24, the blood must either bedosed, within the range of from about 1 nano gram to 100 micrograms perml of blood, with relatively small amounts of activated antibody, so asnot to shock or overload the patient's biological blood filtrationsystem, or the treated blood must be filtered or centrifuged prior toits return to the patient.

Regardless of which photoactivated agent is employed in the invention orat what rate it is administered the burden placed upon the body's organsystem can be further alleviated, by utilizing in conjunction with thepresent system, a continuous centrifuge 44 (or other filtration system),which device serves several functions.

It is to be noted that continuous centrifuges of the type here utilized,have been long employed in blood flow processing systems commerciallyavailable from several manufacturers, including Haemonetics Corporationof Braintree, Mass., and the IBM Corporation, Medical Products Division,of Monsey, N.Y. In the prior art systems in which such devices have beenutilized all elements of FIG. 1 have been present, with the singularlyimportant exception of the irradiation station 24. The function of thecontinuous centrifuge in such prior art systems has been one ofseparating excess lymphocytes or other blood components of interest.Where so used, a detriment of such system was the inefficiency of same,i.e. the centrifuging process can at best remove about 40 to 50% of thelymphocytes, and unfortunately also removes components which are in factdesired to be retained.

In the system 10 of the present invention, two functions can beperformed by the continuous centrifuge 44. One of these, is removal oflymphocytes or other complexed blood constituents, as previouslydiscussed. Because the present invention in its psoralens and cortisonetreatment embodiments relies primarily on impairment of function of thelymphocytes to ultimately reduce the functioning population of same, thecentrifuge 44 need not be relied upon to the extent that same has beenin the aforementioned prior art arrangements. From a mechanicalviewpoint, this implies that one need not work as close to the specificgravity interface between the lymphocyte fraction of the blood and thedesirable fractions of the blood which one seeks to retain. Thus one canavoid undue separation of those desired fractions of the whole blood.

In the embodiments of the invention employing photoactivated antibodies,the antibody complexes formed will be easily separated from the otherdesirable blood fractions, whether by filtration or in the depictedcentrifuge type device 44.

The continuous centrifuge 44, may further be utilized for an additionalimportant purpose. In particular, some or virtually all of the bloodplasma may be removed at 46 and replaced with fresh plasma at 48. Thiswashing technique enables one to effectively withdraw the excessphotoactive chemical agent compounds which may be present in the bloodplasma, replacing the plasma at 46 with isotonic fluid free of the same.Thus, when the blood is returned to the subject at 14, it issubstantially free of any excess chemical agent, i.e. other than thosewhich combined with the treated blood constituent in the manner desired.

It should also be reemphasized that while the preferred mode ofpracticing the present invention, as illustrated in FIG. 1, contemplatesa continuous operation, the blood treatment pursuant to the inventioncan be effected by batched techniques. Thus for example a distinct,fixed quantity of blood may initially be withdrawn from the subject.Such quantity or batch, may already have present therein the desiredquantities of dissolved photoactive chemical agent, i.e. by prioradministration to the patient; or the same agent may be admixedexternally with the withdrawn blood. The said blood batch bearing thedesired agent may then be provided to an irradiation station, where thedesired quantity of UV energy is rendered incident upon same. Duringthis process the batch of blood can be flowed through the station aspreviously discussed, or if the quantity of blood is appropriate and theblood treatment chamber 28 of appropriate dimensions, the batch cansimply be treated under static conditions until the desired energy hasbeen dissipated. Thereafter, the treated blood is taken from theirradiation station, and either centrifuged as above discussed, ordirectly returned to the subject.

The following additional chemical agents are known to interact withintact cells following exposure to UV and visible light. These agentsmay also be used in the system of this invention.

1. Ethidium and acridines (Yielding K. L. and Yielding L. W.:Photoaffinity labeling of DNA. Annals of N.Y. Acad. Sci. 346:368-378,1980).--Also adriamycin, daunomycin, rubidazone.

2. Sulfonamides, sulfonylureas, phenothiazines, tetracyclines, coal tarderivatives, anthracene, pyridine, phenanthrene (Kornhauser A: Molecularaspects of phototoxicity. Annals of N.Y. Acad. Sci.346:398-414, 1980).

3. Specifically reactive antibodies (Richard F. F. and Lifter J.:Photoaffinity probes in the antibody combining region. Annals of N.Y.Acad. Sci. 346:78-89, 1980).

While the present invention has been particularly described in terms ofspecific embodiments thereof, it will be understood in view of thepresent disclosure, that numerous variations upon the invention are nowenabled to those skilled in the art, which variations yet reside withinthe scope of the present invention. Accordingly, the invention is to bebroadly construed, and limited only by the scope and spirit of theclaims now appended hereto.

What is claimed is:
 1. A method for reducing the functioning populationof a nucleated cell in the blood supply of a human subject, comprisingthe steps of:withdrawing blood from said subject, and irradiating saidwithdrawn blood with UV radiation in the presence of a dissolvedphotoactivated antibody specific for the nucleated blood cell andcapable when activated by said UV radiation of forming photoadducts withthe nucleated blood cell, to thereby effect chemical bonding betweensaid photoactivated antibody and the nucleated cells, thereby creatingirreversible complexes of said antibody and said nucleated blood cells;and returning the irradiated blood to said subject.
 2. A method inaccordance with claim 1 wherein the nucleated blood cells arelymphocytes.
 3. A method in accordance with claim 1 or 2 wherein saidwithdrawn blood is formed into an extracorporeal flowing stream, passedthrough a treatment station whereat said irradiation is effected andreturned to said subject in a continuous operation.
 4. A method inaccordance with claim 2 including the further step of separating atleast portions of said photoactivated antibody-lymphocyte complexesbefore returning said blood to said subject.
 5. A method in accordancewith claim 2 including the further step of separating at least portionsof said photoactivated antibody-lymphocyte complexes before returningsaid blood to said subject, by passing said bloodstream through afiltration system or a continuous flow centrifuge.
 6. A method inaccordance of claim 5, wherein excess quantities of said photoactivatedantibody are removed from the extracorporeal stream by withdrawing bloodplasma at said centrifuge and replacing same with fresh photoactivatedantibody-free plasma.
 7. A method in accordance with claim 1 whereinsaid blood is irradiated with photoenergy in the UVA wavelength range,and at a radiation dose level of from about 0.1 to 100 joules/cm².
 8. Amethod in accordance with claim 1 wherein said photoactivated antibodyis dissolved in said blood by mixing same with said blood subsequent towithdrawing the blood from said subject.
 9. A method in accordance withclaim 1 wherein said photoactivated antibody is dissolved in said bloodby administering said photoactivated antibody to said subject.
 10. Amethod for reducing the functioning population of an immunogenicchemical in the blood supply of a human subject, comprising the stepsof:withdrawing blood from said subject, and irradiating said withdrawnblood with UV radiation in the presence of a dissolved photoactivatedantibody specific for the immunogenic chemical and capable whenactivated by said UV radiation of forming photoadducts with saidimmunogenic chemical, to thereby effect chemical bonding between saidphotoactivated antibody and said immunogenic chemical, thereby creatingirreversible complexes of said antibody and said immunogenic chemical;and returning the irradiated blood to said subject.
 11. A method inaccordance with claim 10 wherein the immunogenic chemical is anundesirable natural antibody of the human subject.
 12. A method inaccordance with claim 10 or 11 wherein said withdrawn blood is formedinto an extracorporeal flowing stream, passed through a treatmentstation whereat said irradiation is effected and returned to saidsubject in a continuous operation.
 13. A method in accordance with claim11 including the further step of separating at least portions of saidphotoactivated antibody-undesirable natural antibody complexes beforereturning said blood to said subject.
 14. A method in accordance withclaim 11 including the further step of separating at least portions ofsaid photoactivated antibody-undesirable natural antibody complexesbefore returning said blood to said subject, by passing said bloodstreamthrough a filtration system or a continuous flow centrifuge.
 15. Amethod in accordance with claim 14 wherein excess quantities of saidphotoactivated antibody are removed from the extracorporeal stream bywithdawing blood plasma at said centrifuge and replacing same with freshphotoactivated antibody-free plasma.
 16. A method in accordance withclaim 10 wherein said blood is irradiated with photoenergy in the UVAwavelength range, and at a radiation dose level of from about 0.1 to 100joules/cm².
 17. A method in accordance with claim 10 wherein saidphotoactivated antibody is dissolved in said blood by mixing same withsaid blood subsequent to withdrawing the blood from said subject.
 18. Amethod in accordance with claim 10 wherein said photoactivated antibodyis dissolved in said blood by administering said photoactivated antibodyto said subject.
 19. A method in accordance with claim 1 or 10 whereinthe photoactivated chemical has an azide or diazo moiety contributing toits photoactivity.